Restor Dent Endod.  2023 May;48(2):e18. 10.5395/rde.2023.48.e18.

Effects of CTHRC1 on odontogenic differentiation and angiogenesis in human dental pulp stem cells

• Kim Js
• Lee BN
• Chang HS
• Hwang IN
• Oh WM
• Hwang YC

• KMID: 2548221
• DOI: http://doi.org/10.5395/rde.2023.48.e18

Abstract

Objectives
This study aimed to determine whether collagen triple helix repeat containing-1 (CTHRC1), which is involved in vascular remodeling and bone formation, can stimulate odontogenic differentiation and angiogenesis when administered to human dental pulp stem cells (hDPSCs).
Materials and Methods
The viability of hDPSCs upon exposure to CTHRC1 was assessed with the WST-1 assay. CTHRC1 doses of 5, 10, and 20 µg/mL were administered to hDPSCs. Reverse-transcription polymerase reaction was used to detect dentin sialophosphoprotein, dentin matrix protein 1, vascular endothelial growth factor, and fibroblast growth factor 2. The formation of mineralization nodules was evaluated using Alizarin red. A scratch wound assay was conducted to evaluate the effect of CTHRC1 on cell migration. Data were analyzed using 1-way analysis of variance followed by the Tukey post hoc test. The threshold for statistical significance was set at p < 0.05.
Results
CTHRC1 doses of 5, 10, and 20 µg/mL had no significant effect on the viability of hDPSCs. Mineralized nodules were formed and odontogenic markers were upregulated, indicating that CTHRC1 promoted odontogenic differentiation. Scratch wound assays demonstrated that CTHRC1 significantly enhanced the migration of hDPSCs.
Conclusions
CTHRC1 promoted odontogenic differentiation and mineralization in hDPSCs.

Keyword

Collagen triple helix repeat containing-1; Odontogenic; Mineralization; Dental pulp stem cell

Figure

• 

  Figure 1 The effect of CTHRC1 on the viability of hDPSCs was measured using the WST-1 assay. Cells were incubated with increasing concentrations of CTHRC1 (0, 5, 10, and 20 µg/mL) for 48 hours. Cell viability was significantly lower in the group that was treated with 20 µg/mL CTHRC1 than in the control group (p < 0.05).CTHRC1, collagen triple helix repeat containing 1; hDPSCs, human dental pulp stem cells.

• 

  Figure 2 The effects of CTHRC1 on odontogenic differentiation and angiogenesis in hDPSCs. DSPP, DMP-1, FGF-2, and VEGF expression determined using real-time-polymerase chain reaction. Higher mRNA expression of DSPP and DMP-1 was displayed in hDPSCs treated with 10 µg/mL CTHRC1 in a 10-day culture. However, there was no statistically significant difference.CTHRC1, collagen triple helix repeat containing 1; hDPSCs, human dental pulp stem cells; DSPP, dentin sialophosphoprotein; DMP-1, dentin matrix protein 1; FGF-2, fibroblast growth factor 2; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Figure 3 Results of Alizarin red S staining (21 days). (A) Cells were treated with or without CTHRC1 (5, 10, or 20 µg/mL) in OM medium (50 mg/mL ascorbic acid, 10 mmol/L β-glycerophosphate). (B) The formation of mineralized nodules and calcium deposits significantly increased at 5 and 20 µg/mL concentrations of CTHRC1 (p < 0.05).CTHRC1, collagen triple helix repeat containing 1.

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  Figure 4 CTHRC1 promotes cell migration of hDPSCs. (A) Scratch wound healing assays were performed in hDPSCs treated with CTHRC1. Representative microscopic views at 0 and 24 hours are shown. (B) The quantitative analysis of the decreased gap area after 24 hours shows a significant difference. Different capital letters denote significant differences among treatment groups at the same time. Different small letters denote significant differences in each group over time from 0 to 24 hours (p < 0.05).CTHRC1, collagen triple helix repeat containing 1; hDPSCs, human dental pulp stem cells.

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